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. 2003 Aug 1;22(15):3910-8.
doi: 10.1093/emboj/cdg370.

Essential role of RelA Ser311 phosphorylation by zetaPKC in NF-kappaB transcriptional activation

Angeles Duran  1 , María T Diaz-Meco, Jorge Moscat
Affiliations

Essential role of RelA Ser311 phosphorylation by zetaPKC in NF-kappaB transcriptional activation

Angeles Duran et al. EMBO J. .

Abstract

The activation of the transcription factor NF-kappaB is central to the control of the cellular response triggered by many stimuli. Once released from the inhibitory molecule IkappaB, NF-kappaB is translocated to the nucleus, and it has to be phosphorylated to activate transcription. In zeta protein kinase C (PKC)-deficient cells, NF-kappaB is transcriptionally inactive and the phosphorylation of the RelA subunit in response to tumor necrosis factor (TNF-alpha) is severely impaired. In vitro assays showed that zetaPKC directly phosphorylates RelA. Here we demonstrate that Ser311 accounts for zetaPKC phosphorylation of RelA and that this site is phosphorylated in vivo in response to TNF-alpha. Also, an inactivating mutation of that residue severely impairs RelA transcriptional activity, blocks its anti-apoptotic function and abrogates the interaction of RelA with the co-activator CBP as well as its recruitment, and that of RNA polymerase II (Pol II) with the interleukin-6 (IL-6) promoter. The interaction of endogenous CBP with endogenous RelA is inhibited in zetaPKC-/- cells, as well as the binding of Pol II to the IL-6 promoter. These results demonstrate the mechanism whereby zetaPKC regulates NF-kappaB activation in vivo.

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Figures

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Fig. 1. Mapping of the ζPKC phosphorylation site in RelA. (A) Recombinant RelA RHD was phosphorylated in vitro with recombinant baculovirus ζPKC. The RelA tryptic phosphopeptides were separated by electrophoresis followed by ascending chromatography. (B) Putative ζPKC phosphorylation sites in RelA as predicted by the Scansite program. (C) Recombinant RelA RHD either wild-type (RelA, left and right panels) or mutated (RelAT308A, left panel; RelAS311A, right panel) was phosphorylated in vitro by recombinant ζPKC. (D) Recombinant RelA RHD either wild-type or S311A mutant was phosphorylated in vitro with pure PKA and the tryptic peptides were separated by electrophoresis followed by ascending chromatography as above. (E) Recombinant RelA RHD either wild-type or mutated at Ser311 or Ser276 was phosphorylated in vitro with ζPKC as above. (F) Recombinant RelA wild-type or S311A mutant was incubated in phosphorylation reactions with ζPKC, after which the phosphorylation of Ser311 was determined by immunoblotting with the anti-phospho-RelA(Ser311) antibody. As a negative control, wild-type RelA was incubated in the absence of ζPKC.
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Fig. 2. Phosphorylation of RelA Ser311 in vivo. (A) Control EFs or stably expressing HA-tagged wild-type or S311A mutant RelA were incubated with TNF-α for different times, after which the ectopically expressed RelA was immunoprecipitated with the anti-HA antibody and Ser311 phosphorylation was determined by immunoblotting with the anti-phospho-RelA(Ser311) antibody (upper panel). The lower panel shows a parallel immunoblot with the anti-HA antibody. (B) The immunoprecipitates from the cells that had been treated with TNF-α for 30 min were immunoblotted with the anti-phospho-RelA(Ser311) antibody in the absence or presence of phospho- or dephospho-peptide. (C) Wild-type or ζPKC-deficient EFs were stimulated with TNF-α for different times, after which endogenous RelA was immunoprecipitated and Ser311 phosphorylation was determined by immunoblotting as above.
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Fig. 3. Role of Ser311 in NF-κB transcription. (A) Subconfluent cultures of 293 cells were transfected with either empty plasmid (striped bar) or different amounts of expression vectors for HA-tagged wild-type or S311A mutant RelA along with a κB-dependent reporter and the Renilla control plasmid. Luciferase activity was determined as described in Materials and methods. Results are the mean ± SD of three independent experiments with incubations in duplicate (left panel). The expression levels of wild-type and mutant RelA of one representative experiment were determined by immunoblotting with an anti-HA antibody (right panel). (B) EFs transduced with empty pBabe vector (control) or stably expressing wild-type or S311A mutant RelA were transfected with the κB-luciferase reporter vector along with the control Renilla plasmid, after which they were stimulated for 6 h with different concentrations of TNF-α (upper left panel) or the agonistic anti-lymphotoxin-β receptor antibody ACH6 (lower panel). Luciferase activity was determined as above. Expression levels of endogenous and ectopically expressed RelA proteins were determined by immunoblotting with an anti-RelA antibody (upper right panel).
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Fig. 4. Lack of effect of S311A mutation on NF-κB nuclear activity. (A) Nuclear extracts from cells expressing wild-type or the S311A mutant that had been stimulated or not with TNF-α for 1 h were analyzed by EMSA using a κB probe. These nuclear extracts were also pre-incubated with anti-RelA or anti-HA antibodies to determine the contribution of the ectopically expressed RelA proteins to the NF-κB complex. (B) RelA–/– cells that had been transduced with wild-type or mutant RelA proteins were stimulated or not with TNF-α for 1 h and nuclear extracts analyzed by EMSA as above (upper panel), or total cell extracts were analyzed by immunoblotting with anti-HA antibody.
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Fig. 5. Control of IL-6 synthesis by ζPKC and RelA Ser311 phosphorylation. (A) EFs from wild-type (WT) or ζPKC-deficient mice (KO) were stimulated with different concentrations of TNF-α for 24 h, after which IL-6 production was determined by ELISA. (B) IL-6 production was also determined in EFs stably expressing WT or the S311A RelA mutant. Results are the mean ± SD of two independent experiments with incubations in duplicate.
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Fig. 6. ζPKC is required for RelA-induced IL-6 synthesis. Either wild-type or ζPKC-deficient EFs were transfected with wild-type RelA or the different mutants, after which cells were stimulated or not with TNF-α, and IL-6 synthesis was determined as above (upper panel). Immunoblot analysis with anti-RelA antibody shows the expression levels of the transfected and endogenous RelA protein.
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Fig. 7. Role of Ser311 in NF-κB-induced survival. Control EFs or stably expressing wild-type or S311A RelA mutant were incubated in the absence or presence of TNF-α plus cycloheximide for 72 (upper panel) or 24 h (lower panel), after which the percentage of cells undergoing apoptosis was determined by TUNEL analysis. Results are the mean ± SD of two independent experiments with incubations in duplicate. P-values were calculated according to Student’s t-test. *P < 0.01; **P < 0.001.
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Fig. 8. Role of Ser311 in CBP-enhanced NF-κB transcriptional activity. EFs stably expressing wild-type or the S311A RelA mutant were transfected with the κB-dependent reporter and the Renilla control plasmid, along with increasing amounts of a CBP expression plasmid, after which they were either untreated or stimulated with TNF-α for 6 h and the luciferase activity was determined as above (A). Immublot analysis with an anti-CBP antibody shows the levels of expression of the transfected CBP (B).
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Fig. 9. RelA–CBP interaction and promoter recruitment. (A) EFs stably expressing wild-type or the S311A RelA mutant were stimulated with TNF-α for 1 h, after which cell extracts were immunoprecipitated with an anti-HA antibody and the immunoprecipitates were analyzed by immunoblotting with an anti-CBP or anti-HA antibody (left panel). Wild-type or ζPKC-deficient EFs, or ζPKC-deficient EFs in which HA-ζPKC was ectopically expressed were stimulated with TNF-α for 1 h, after which endogenous RelA was immunoprecipitated and the associated endogenous CBP was determined by immunoblotting (right panel). (B) Cells as above were stimulated or not with TNF-α for 90 min, after which cross-linked chromatin was immunoprecipitated with anti-CBP or anti-RNA Pol II antibodies, and the IL-6 promoter was detected by PCR in these samples using promoter-specific primers. (C) Either wild-type or ζPKC–/– EFs were stimulated with TNF-α for 90 min and the ChIP analysis was performed to determine Pol II recruitment as above. Input shows the starting chromatin extracts.
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